Iron-Chelation Treatment by Novel Thiosemicarbazone Targets Major Signaling Pathways in Neuroblastoma

Despite constant advances in the field of pediatric oncology, the survival rate of high-risk neuroblastoma patients remains poor. The molecular and genetic features of neuroblastoma, such as MYCN amplification and stemness status, have established themselves not only as potent prognostic and predictive factors but also as intriguing targets for personalized therapy. Novel thiosemicarbazones target both total level and activity of a number of proteins involved in some of the most important signaling pathways in neuroblastoma. In this study, we found that di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) potently decreases N-MYC in MYCN-amplified and c-MYC in MYCN-nonamplified neuroblastoma cell lines. Furthermore, DpC succeeded in downregulating total EGFR and phosphorylation of its most prominent tyrosine residues through the involvement of NDRG1, a positive prognostic marker in neuroblastoma, which was markedly upregulated after thiosemicarbazone treatment. These findings could provide useful knowledge for the treatment of MYC-driven neuroblastomas that are unresponsive to conventional therapies.

YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity

The amino-terminal cytoplasmic domains of paramyxovirus fusion glycoproteins include trafficking signals that influence protein processing and cell surface expression. This study clarified that tyrosine residues at different positions in the YLMY motif in the cytoplasmic region of the F protein regulate F protein transportation, thereby affecting viral replication and pathogenicity.

Protective Effect of Dinitrosyl Iron Complexes Bound with Hemoglobin on Oxidative Modification by Peroxynitrite

Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide (•NO) in an organism. They are able not only to deposit and transport •NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia.

Computational Screening for the Anticancer Potential of Seed-Derived Antioxidant Peptides: A Cheminformatic Approach

Some seed-derived antioxidant peptides are known to regulate cellular modulators of ROS production, including those proposed to be promising targets of anticancer therapy. Nevertheless, research in this direction is relatively slow owing to the inevitable time-consuming nature of wet-lab experimentations. To help expedite such explorations, we performed structure-based virtual screening on seed-derived antioxidant peptides in the literature for anticancer potential. The ability of the peptides to interact with myeloperoxidase, xanthine oxidase, Keap1, and p47phox was examined. We generated a virtual library of 677 peptides based on a database and literature search. Screening for anticancer potential, non-toxicity, non-allergenicity, non-hemolyticity narrowed down the collection to five candidates. Molecular docking found LYSPH as the most promising in targeting myeloperoxidase, xanthine oxidase, and Keap1, whereas PSYLNTPLL was the best candidate to bind stably to key residues in p47phox. Stability of the four peptide-target complexes was supported by molecular dynamics simulation. LYSPH and PSYLNTPLL were predicted to have cell- and blood-brain barrier penetrating potential, although intolerant to gastrointestinal digestion. Computational alanine scanning found tyrosine residues in both peptides as crucial to stable binding to the targets. Overall, LYSPH and PSYLNTPLL are two potential anticancer peptides that deserve deeper exploration in future.

Src activates retrograde membrane traffic through phosphorylation of GBF1

The Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP Exchange factor (GEF) GBF1 and small GTPase Arf1. Here we show that Src induces the formation of tubular transport carriers containing GALNTs. The kinase phosphorylates GBF1 on 10 tyrosine residues; two of them, Y876 and Y898 are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes GBF1 binding to the GTPase; molecular modeling suggests partial melting of the Sec7 domain and intramolecular rearrangement. GBF1 mutants defective for these rearrangements prevent binding, carrier formation and GALNTs relocation, while phosphomimetic GBF1 mutants induce tubules. In sum, Src promotes GALNTs relocation by promoting GBF1 binding to Arf1. Based on residue conservation, similar regulation of GEF-Arf complexes by tyrosine phosphorylation could be a conserved and wide-spread mechanism.

Activation of the STAT1-BCL-2/MCL-1 Axis in Leukemic Cells Carrying a SPAG9-JAK2 Fusion

[Background and aim of this study] Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a distinct subtype of B-ALL with poor prognosis. JAK2 related fusion genes have been identified in this subtype, especially in adolescent and young adults (AYA). Previously, we identified SPAG9-JAK2 fusion gene in 14-year-old boy with Ph-like ALL (Kawamura M, et al. Genes Chromosomes Cancer. 2015). In this study, we performed functional analysis of the SPAG9-JAK2 fusion protein, and evaluated the efficacy of treatment with a JAK inhibitor against cells carrying the fusion. In addition, we assessed therapeutic options other than JAK2 inhibition following comprehensive molecular analysis. [Materials and Methods] Full length of SPAG9-JAK2 cDNA was cloned into retroviral construct with Tet-On system. Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing SPAG9-JAK2 (Ba/F3-SPAG9-JAK2) under doxycycline (DOX) dependent manner. Ba/F3-SPAG9-JAK2 were analyzed whether IL-3 independent growth was achieved. Aberrant activation of JAK-STAT pathway achieved by SPAG9-JAK2 was evaluated by western blot. To clarify whether the tyrosine residues of JAK2 in this fusion protein were critical for IL-3-independent proliferation, Ba/F3 cells expressing SPAG9-JAK2 mutants (SPAG9-JAK2 mut) in which both tyrosine residues of JAK2 were replaced with phenylalanine was established. Gene expression analysis using Mouse Genome 430 2.0 Array was performed for comprehensive analysis of gene expression profile related to SPAG9-JAK2. Sensitivity of Ba/F3-SPAG9-JAK2 to ruxolitinib (a JAK inhibitor) was tested in cytotoxic assay and in xenograft model established using Ba/F3-SPAG9-JAK2 cells. [Results and discussions] The expression of SPAG9-JAK2 in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-SPAG9-JAK2 proliferated without IL-3 in contrast to Ba/F3 cells (p<0.01), suggesting SPAG9-JAK2 had the proliferation activity. Western blot revealed that constitutive phosphorylation of tyrosine residue of SPAG9-JAK2, STAT3/STAT5, suggesting constitutive activation of JAK2-STAT3/STAT5 pathway. SPAG9-JAK2 mut abolished IL-3 independence (p<0.01), but had no influence on STAT3/STAT5 phosphorylation levels detected by western blot. Gene expression analysis revealed that Stat1 was significantly up-regulated in Ba/F3-SPAG9-JAK2 cells compared with mock Ba/F3 cells (fold change 8.04 with p < 0.01) [Fig. 1], confirmed by western blot. STAT1 was also phosphorylated in Ba/F3-SPAG9-JAK2 but not SPAG9-JAK2 mut cells detected by western blot [Fig. 2], suggesting that STAT1 is a key mediator for SPAG9-JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9-JAK2, but not SPAG9-JAK2 mut confirmed by western blot [Fig. 3]. Ruxolitinib abrogated Ba/F3-SPAG9-JAK2-mediated proliferation in vitro (p<0.01), with an 50% inhibitory concentration (IC50) value of 65.9 ± 9.8 nM, causing decrease of JAK2, STAT1/STAT3/STAT5 phospholyration in western blot and apoptosis in annexin V/PI staining. Ruxolitinib prolonged survival time of xenotransplanted mice (p = 0.0213), however, the proliferation of leukemic cells in mouse bone marrow was not suppressed by ruxolitinib. Ba/F3-SPAG9-JAK2 cells showed a dose-dependent response for venetoclax (a BCL-2 inhibitor) with IC50 2.57 ± 1.11 µM and AZD5991 (an MCL-1 inhibitor) with IC50 6.76 ± 3.31 µM. Treatment of Ba/F3-SPAG9-JAK2 cells with a combination of ruxolitinib and venetoclax or AZD5991 resulted in a significant reduction in the IC50 of ruxolitinib (p<0.01) [Fig. 4, 5], with a combined index (CI) value of 0.61 or 0.92, indicating a moderately or weak synergistic effect in vitro. [Conclusion] SPAG9-JAK2 promotes cell proliferation and that tyrosine phosphorylation of the JAK2 kinase domain is critical for IL-3-independent cell growth. Ruxolitinib shows sufficient cytotoxic effects against Ba/F3-SPAG9-JAK2 cells in vitro, but is only partially effective in vivo. Activation of the JAK2-STAT1-BCL-2/MCL-1 axis contributes to aberrant growth promotion by SPAG9-JAK2. BCL-2 or MCL-1 inhibitors in combination with ruxolitinib shows efficacy against Ph-like ALL carrying the SPAG9-JAK2 fusion in vitro. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The bacterial tyrosine kinase system CpsBCD governs the length of capsule polymers

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae. Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse–chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

Self-Assembled Silk Fibroin-Based Aggregates for Delivery of Camptothecin

A water-soluble hydrolysate of silk fibroin (SF) (~30 kDa) was esterified with tocopherol, ergocalciferol, and testosterone to form SF aggregates for the controlled delivery of the anticancer drug camptothecin (CPT). Elemental analysis and 1H NMR spectroscopy showed a degree of substitution (DS) on SF of 0.4 to 3.8 mol %. Yields of 58 to 71% on vitamins- and testosterone-grafted SF conjugates were achieved. CPT was efficiently incorporated into the lipophilic core of SF aggregates using a dialysis–precipitation method, achieving drug contents of 6.3–8.5 wt %. FTIR spectra and DSC thermograms showed that tocopherol- and testosterone-grafted SF conjugates predominantly adopted a β-sheet conformation. After the esterification of tyrosine residues on SF chains with the vitamin or testosterone, the hydrodynamic diameters almost doubled or tripled that of SF. The zeta potential values after esterification increased to about −30 mV, which favors the stability of aggregates in aqueous medium. Controlled and almost quantitative release of CPT was achieved after 6 days in PBS at 37 °C, with almost linear release during the first 8 h. MCF-7 cancer cells exhibited good uptake of CPT-loaded SF aggregates after 6 h, causing cell death and cell cycle arrest in the G2/M phase. Substantial uptake of the CPT-loaded aggregates into MCF-7 spheroids was shown after 3 days. Furthermore, all CPT-loaded SF aggregates demonstrated superior toxicity to MCF-7 spheroids compared with parent CPT. Blank SF aggregates induced no hemolysis at pH 6.2 and 7.4, while CPT-loaded SF aggregates provoked hemolysis at pH 6.2 but not at pH 7.4. In contrast, parent CPT caused hemolysis at both pH tested. Therefore, CPT-loaded SF aggregates are promising candidates for chemotherapy.

The structure of natively iodinated bovine thyroglobulin

Thyroglobulin is a homodimeric glycoprotein that is essential for the generation of thyroid hormones in vertebrates. Upon secretion into the lumen of follicles in the thyroid gland, tyrosine residues within the protein become iodinated to produce monoiodotyrosine (MIT) and diiodotyrosine (DIT). A subset of evolutionarily conserved pairs of DIT (and MIT) residues can then engage in oxidative coupling reactions that yield either thyroxine (T4; produced from coupling of a DIT `acceptor' with a DIT `donor') or triiodothyronine (T3; produced from coupling of a DIT acceptor with an MIT donor). Although multiple iodotyrosine residues have been identified as potential donors and acceptors, the specificity and structural context of the pairings (i.e. which donor is paired with which acceptor) have remained unclear. Here, single-particle cryogenic electron microscopy (cryoEM) was used to generate a high-resolution reconstruction of bovine thyroglobulin (2.3 Å resolution in the core region and 2.6 Å overall), allowing the structural characterization of two post-reaction acceptor–donor pairs as well as tyrosine residues modified as MIT and DIT. A substantial spatial separation between donor Tyr149 and acceptor Tyr24 was observed, suggesting that for thyroxine synthesis significant peptide motion is required for coupling at the evolutionarily conserved thyroglobulin amino-terminus.

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton’s tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1β release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.